This invention is directed to the use of phosphotyrosine phosphatase inhibitors and tyrosine kinase activators for controlling cellular proliferation, particularly proliferation of lymphocytes.
Tyrosine phosphorylation is known to play an essential role in the control of lymphocyte function. This control is exerted by a network of tyrosine kinases and phosphotyrosine phosphatases.
Apoptosis is a pattern of programmed cell death that involves a breakup of the cellular DNA and can be recognized by electrophoresis. Apoptosis is an important lymphocyte response believed to play a prominent role in T and B lymphocyte maturation in the thymus (T cells) and in germinal centers (B cells), an example being the process of negative selection of self-reactive cells (J. J. Cohen et al., "Apoptosis and Programmed Cell Death in Immunity," Annu. Rev. Immunol. 10:267-293 (1992); D. R. Green et al., "Activation-Induced Apoptosis in Lymphoid Systems," Sem. Immunol. 4:379-388 (1992)).
Three different means of inducing apoptosis in lymphocytes all appear to require tyrosine phosphorylation. First, stimulation of the antigen receptor can lead to lymphocyte apoptosis. In the case of B cells, treatment of immature B cell lymphomas, but not mature B cells, with soluble anti-Ig antibodies often induces apoptosis (L. E. Benhamou et al., "Anti-Immunoglobulins Induce Death by Apoptosis in WEHI-231 B Lymphoma Cells," Eur. J. Immunol. 20:1405-1407 (1990); J. Hasbold & G. G. B. Klaus, "Anti-Immunoglobulin Antibodies Induce Apoptosis in Immature B Cell Lymphomas," Eur. J. Immunol. 20:1685-1690 (1990)). Signaling by anti-Ig antibodies requires tyrosine phosphorylation as an early and essential step (A. L. DeFranco, "Structure and Function of the B Cell Antigen Receptor," Annu. Rev. Cell Biol. 9:377-410 (1993)).
In immature B cells, stimulation of sIgM (surface immunoglobulin M) by either antigen or anti-immunoglobulin antibodies activates the cells (G. J. V. Nossal, Annu. Rev. Immunol. 1:33-62 (1983)). Stimulation of sIg (surface immunoglobulin) in B cells induces tyrosine phosphorylation (M. R. Gold et al., Nature 345:810-813 (1990); M. A. Campbell & B. M. Sefton, EMBO J. 9:2125-2131 (1990), which is essential for productive sIg signaling (P. J. L. Lane et al., J. Immunol. 146:715-722 (1991)).
Furthermore, specific tyrosine kinases appear to be involved in the B cell signaling that leads to apoptosis or growth arrest. For example, as a result of sIg stimulation, Src family kinases are activated (A. L. Burkhardt et al., Proc. Natl. Acad. Sci. USA 88:7410-7414 (1991)). Additionally, expression of the Src family tyrosine kinase Blk was found to be essential in B cell lymphomas where sIgM stimulation leads to growth arrest and apoptosis (X. R. Yao & D. W. Scott, "Expression of Protein Tyrosine Kinases in the Ig Complex of Anti-.mu.-Sensitive and Anti-.mu.-Resistant B Cell Lymphomas: Role of the p55.sup.blk Kinase in Signaling Growth Arrest and Apoptosis," Immunol. Rev. 132:163-186 (1993)). Similarly, the expression of the Blk tyrosine kinase has been found to be necessary for antigen receptor induced apoptosis in CH31 lymphoma cells (X. R. Yao & D. W. Scott, "Antisense Oligodeoxynucleotides to the blk Tyrosine Kinase Prevent Anti-.mu.-Chain-Mediated Growth Inhibition and Apoptosis in a B Cell Lymphoma," Proc. Natl. Acad. Sci. USA 90:7946-7950 (1993)), wherein the Lyn tyrosine kinase has been shown to be necessary for antigen receptor induced growth arrest in both human and murine B cell lymphoma lines (R. H. Scheuermann et al., "Lyn Tyrosine Kinase Signals Cell Cycle Arrest but Not Apoptosis in B-Lineage Lymphoma Cells," Proc. Natl. Acad. Sci. USA 91:4048-4052 (1994)).
Thus, on sIgM stimulation, tyrosine kinases such as Blk phosphorylate one or more proteins on tyrosine residues, and once phosphorylated, these proteins are then able to induce apoptosis. However, it has also been shown that the abundant phosphotyrosine phosphatase CD45 is required for sIg signal transduction (L. B. Justement et al., Science 252:1839-1842 (1991)).
The correlation of the ability of monoclonal anti-idiotypic antibodies to induce tyrosine phosphorylation signaling and their ability to produce lymphoma regression in human patients also supports the role of tyrosine phosphorylation in these processes (W. M. J. Vuist et al., "Lymphoma Regression Induced by Monoclonal Anti-Idiotypic Antibodies Correlates with Their Ability to Induce Ig Signal Transduction and Is Not Prevented by Tumor Expression of High Levels of Bcl-2 Protein," Blood 83:899-906 (1994)). In the case of T cells, the tyrosine kinase inhibitor herbimycin A prevented superantigen induced cell death that otherwise resulted from cross-linking multiple antigen receptors (N. K. Damle et al., "Activation with Superantigens Induces Programmed Death in Antigen-Primed CD4+ Class II+ Major Histocompatibility Complex T Lymphocytes Via a CD11a/CD18-Dependent Mechanism," Eur. J. Immunol. 23:1513-1522 (1993)).
A second example of the role of tyrosine phosphorylation in lymphocyte apoptosis is the case of Fas induced cell death in T cells. Crosslinking of Fas antigen on Jurkat T cell leukemia cells with anti-Fas antibodies induces both apoptosis and activation of tyrosine kinases leading to cellular tyrosine phosphorylation (C. M. Eischen et al., "Tyrosine Kinase Activation Provides an Early and Requisite Signal for Fas-Induced Apoptosis," J. Immunol. 153:1947-1954 (1994)). Herbimycin A blocked both the Fas induced tyrosine phosphorylation and death in these cells, demonstrating the essential role of the tyrosine kinases.
Apoptosis induced by ionizing radiation provides a third example. Ionizing radiation causes tyrosine kinase activation in human B cell lymphocyte precursors at therapeutically relevant doses that lead to growth arrest in apoptotic cell death (F. M. Uckun et al., "Ionizing Radiation Stimulates Unidentified Tyrosine-Specific Protein Kinases in Human B-Lymphocyte Precursors Triggering Apoptosis and Clonogenic Cell Death," Proc. Natl. Acad. Sci. USA 89:9005-9009 (1992)). Ionizing radiation is standard therapy for B cell malignancies such as leukemias and lymphomas. Tyrosine kinase inhibitors blocked the radiation-induced tyrosine phosphorylation and apoptosis. The phosphotyrosine phosphatase (PTP) inhibitor vanadate, which when used alone had little effect, greatly augmented the radiation-induced tyrosine phosphorylation and cell death (F. M. Uckun et al. (1992), supra). The activation of tyrosine kinases by ionizing radiation was essential for the induction of apoptosis because the tyrosine kinase inhibitors genistein and herbimycin A blocked the effects of the radiation. These results indicate that not only is tyrosine kinase activation essential for radiation induced apoptosis in B cells, but also suggest that phosphatases can act to limit these responses. The state of lymphocyte development also appears to be important in apoptotic responses since immature B cell lines frequently respond to Ig antibody treatment with apoptosis or growth arrest, whereas mature B cells respond by proliferation (R. H. Scheuermann et al. (1994), supra; X. R. Yao & D. W. Scott, Immunol. Rev. (1993), supra; G. J. V. Nossal, "Cellular and Molecular Mechanisms of B Lymphocyte Tolerance," Adv. Immunol. 52:283-331 (1992)).
Tyrosine kinase activation and the phosphorylation of key signaling molecules such as PLC.gamma. are early and essential steps in lymphocyte antigen receptor signal transduction (A. Weiss & D. R. Littman, Cell 76: 263-274 (1994)). Src-family kinases are believed to act first, followed by the Syk family kinases Syk in B cells (S. J. Saouaf et al., Proc. Natl. Acad. Sci. USA 91: 9524-9528 (1994)) and ZAP-70 in T cells (M. Iwashima et al., Science 263: 1136-1139 (1994)). Phosphotyrosine phosphatases (PTP) provide both positive and negative regulation of these signals. CD45 is essential for signaling in both T and B cells, acting at least in part by dephosphorylating the negative regulatory C-terminal phosphorylation site in Src-family kinases, permitting them to become activated (A. Weiss & D. R. Littman (1994), supra; L. B. Justement et al., Science 252: 1839-1842 (1991)). However, CD45 can also negatively regulate T cell signaling by dephosphorylating specific substrates such as the .zeta. chain of the T cell receptor (T. Furukawa et al., Proc. Natl. Acad. Sci. USA 91: 10928-10932 (1994)). In addition, PTP1C has recently been reported to negatively regulate B cell antigen receptor signals (J. G. Cyster & C. C. Goodnow, Immunity 2: 13-24 (1995)). It is likely that additional PTPs are also involved in regulating lymphocyte signal transduction.
Although mature B cells usually respond to antigen receptor signals by proliferation, immature B cell lines frequently respond by undergoing apoptosis or programmed cell death (X. R. Yao & D. W. Scott, Immunol. Rev. 132: 163-186 (1993); R. H. Scheuermann et al., Proc. Natl. Acad. Sci. USA 91: 4048-4052 (1994); G. J. V. Nossal, Adv. Immunol. 52: 283-331 (1992)). This response permits the process of negative selection that eliminates self-reactive immature B cells. This activation induced B cell death requires specific tyrosine kinases involved in antigen receptor signaling, such as the Blk tyrosine kinase (X. R. Yao & D. W. Scott, Proc. Natl. Acad. Sci. USA 90: 7946-7950 (1993)). Recently, it has been reported that mice deficient in PTP1C more readily eliminate self-reactive B cells (J. G. Cyster & C. C. Goodnow (1995), supra), indicating a role for phosphotyrosine phosphatases in this process. Tyrosine kinase activation has also been found to be essential for the ability of therapeutically relevant doses of ionizing radiation to induce tyrosine phosphorylation and apoptosis in human leukemic B cells (F. M. Uckun et al., Proc. Natl. Acad. Sci. USA 89: 9005-9009 (1992)). Tyrosine kinase inhibitors blocked the radiation-induced tyrosine phosphorylation and apoptosis. The PTP inhibitor vanadate, which when used alone had little effect, greatly augmented the radiation-induced tyrosine phosphorylation and cell death (F. M. Uckun et al. (1992), supra). These results indicate that not only is tyrosine kinase activation essential for radiation induced apoptosis in B cells, but also suggest that phosphatases can act to limit these responses.
Despite these findings, the currently available PTP inhibitors are unsatisfactory for in vivo or clinical use. The PTP inhibitors phenylarsine oxide (PAO) (P. G. Garcia-Morales et al., Proc. Natl. Acad. Sci. USA 87: 9255-9259 (1990); M. C. Fletcher et al., J. Biol. Chem. 268: 23697-23703 (1993)) and pervanadate (J. J. O'Shea et al., Proc. Natl. Acad. Sci. USA 89: 10306-10310 (1993); G. L. Schieven et al., Blood 82: 1212-1220 (1993)) have properties that can limit their utility for biological studies and make them unsatisfactory for clinical use. PAO is a highly toxic molecule that has the potential to react with vicinal sulfhydryl groups on a wide variety of proteins (S. C. Frost & M. D. Lane, J. Biol. Chem. 260: 2646-2652 (1985)). Pervanadate is an unstable compound that can give extraordinary induction of tyrosine phosphorylation in the absence of biological stimulation in all cell types examined (D. Heffetz & Y. Zick, J. Biol. Chem. 264: 10126-10132 (1990); D. Heffetz et al., J. Biol. Chem. 265: 2896-2902 (1990)). Furthermore, the effect of PTP inhibition on B cell receptor signaling has not been addressed. Additionally, it would be highly desirable to be able to manipulate the state of tyrosine phosphorylation, such as by the use of phosphotyrosine phosphatase inhibitors, without the use of ionizing radiation.
In addition to blocking proliferation of malignant B cells or T cells in diseases such as leukemias and lymphomas, in a number of situations it may be desirable to slow the growth and/or differentiation of normal B cells or T cells. Such occasions include organ transplantation, in which the immune response, at least in the short term, must be suppressed. Limited control of the proliferation of B cells may also be desirable in the treatment of autoimmune diseases such as rheumatoid arthritis and lupus erythematosus.
Accordingly, there exists a need for improved methods of controlling proliferation of B cells or T cells in malignant and non-malignant conditions without requiring the use of radiation. Such an approach preferably involves the production of programmed cell death (apoptosis) in susceptible cells. Preferably, such methods should be specific for lymphocytes and not cause neutropenia. Also, such methods should be of value in treating tumors overexpressing one or more members of the EGF receptor family, other receptor tyrosine kinases, or other tyrosine kinases.